Acyl dihydro pyrrole derivatives as hcv inhibitors

ABSTRACT

Novel anti-viral agents of Formula (I)  
                 
wherein: 
         A represents OR 1 , NR 1 R 2 , or R 1  wherein R 1  and R 2  are hydrogen, C 1-6 alkyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl; or R 1  and R 2  together with the nitrogen atom to which they are attached form a 5 or 6 membered saturated cyclic group;    B represents C(O)R 3  wherein R 3  is C 1-6 alkyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl;    D represents C 1-6 alkyl, aryl, heteroaryl or heterocyclyl;    E represents OR 1 , NR 1 R 2 , or R 1  wherein R 1  and R 2  are hydrogen, C 1-6 alkyl, aryl, heteroaryl, arylalkyl or heteroarylalkyl; or R 1  and R 2  together with the nitrogen atom to which they are attached form a 5 or 6 membered saturated cyclic group;    F represents hydrogen, C 1-6 alkyl, aryl or heteroaryl; and    G represents hydrogen, C 1-6 alkyl, heterocyclylalkyl, arylalkyl or heteroarylalkyl; and salts and solvates thereof, processes for their preparation and methods of using them in HCV treatment are provided.

FIELD OF THE INVENTION

The present invention relates to novel acyl dihydro pyrrole derivativesuseful as anti-viral agents. Specifically, the present inventioninvolves novel HCV inhibitors.

BACKGROUND OF THE INVENTION

Infection with HCV is a major cause of human liver disease throughoutthe world. In the US, an estimated 4.5 million Americans are chronicallyinfected with HCV. Although only 30% of acute infections aresymptomatic, greater than 85% of infected individuals develop chronic,persistent infection. Treatment costs for HCV infection have beenestimated at $5.46 billion for the US in 1997. Worldwide over 200million people are estimated to be infected chronically. HCV infectionis responsible for 40-60% of all chronic liver disease and 30% of allliver transplants. Chronic HCV infection accounts for 30% of allcirrhosis, end-stage liver disease, and liver cancer in the U.S. The CDCestimates that the number of deaths due to HCV will minimally increaseto 38,000/year by the year 2010.

Due to the high degree of variability in the viral surface antigens,existence of multiple viral genotypes, and demonstrated specificity ofimmunity, the development of a successful vaccine in the near future isunlikely. Alpha-interferon (alone or in combination with ribavirin) hasbeen widely used since its approval for treatment of chronic HCVinfection. However, adverse side effects are commonly associated withthis treatment: flu-like symptoms, leukopenia, thrombocytopenia,depression from interferon, as well as anemia induced by ribavirin(Lindsay, K. L. (1997) Hepatology 26 (suppl 1):71S-77S). This therapyremains less effective against infections caused by HCV genotype 1(which constitutes ˜75% of all HCV infections in the developed markets)compared to infections caused by the other 5 major HCV genotypes.Unfortunately, only ˜50-80% of the patients respond to this treatment(measured by a reduction in serum HCV RNA levels and normalization ofliver enzymes) and, of those treated, 50-70% relapse within 6 months ofcessation of treatment. Recently, with the introduction of pegylatedinterferon, both initial and sustained response rates have improvedsubstantially, and combination treatment of Peg-IFN with ribavirinconstitutes the gold standard for therapy. However, the side effectsassociated with combination therapy and the impaired response inpatients with genotype 1 present opportunities for improvement in themanagement of this disease.

First identified by molecular cloning in 1989 (Choo, Q-L et al (1989)Science 244:359-362), hepatitis C virus (HCV) is now widely accepted asthe most common causative agent of post-transfusion non A, non-Bhepatitis (NANBH) (Kuo, G et al (1989) Science 244:362-364).

Due to its genome structure and sequence homology, this virus wasassigned as a new genus in the Flaviviridae family. Like the othermembers of the Flaviviridae, such as flaviviruses (e.g. yellow fevervirus and Dengue virus types 1-4) and pestiviruses (e.g. bovine viraldiarrhea virus, border disease virus, and classic swine fever virus)(Choo, Q-L et al (1989) Science 244:359-3; Miller, R. H. and R. H.Purcell (1990) Proc. Natl. Acad. Sci. USA 87:2057-2061), HCV is anenveloped virus containing a single strand RNA molecule of positivepolarity. The HCV genome is approximately 9.6 kilobases (kb) with along, highly conserved, noncapped 5′ nontranslated region (NTR) ofapproximately 340 bases which functions as an internal ribosome entrysite (IRES) (Wang CY et al ‘An RNA pseudoknot is an essential structuralelement of the internal ribosome entry site located within the hepatitisC virus 5′ noncoding region’ RNA—A Publication of the RNA Society;1(5):526-537, July 1995). This element is followed by a region whichencodes a single long open reading frame (ORF) encoding a polypeptide of˜3000 amino acids comprising both the structural and nonstructural viralproteins.

Upon entry into the cytoplasm of the cell, this RNA is directlytranslated into a polypeptide of ˜3000 amino acids comprising both thestructural and nonstructural viral proteins. This large polypeptide issubsequently processed into the individual structural and nonstructuralproteins by a combination of host and virally-encoded proteinases (Rice,C. M. (1996) in B. N. Fields, D. M. Knipe and P. M. Howley (eds)Virology 2^(nd) Edition, p931-960; Raven Press, N.Y.). Following thetermination codon at the end of the long ORF, there is a 3′ NOR whichroughly consists of three regions: an ˜40 base region which is poorlyconserved among various genotypes, a variable lengthpoly(U)/polypyrimidine tract, and a highly conserved 98 base elementalso called the “3′ X-tail” (Kolykhalov, A. et al (1996) J. Virology70:3363-3371; Tanaka, T. et al (1995) Biochem Biophys. Res. Conmmun.215:744-749; Tanaka, T. et al (1996) J. Virology 70:3307-3312; Yamada,N. et al (1996) Virology 223:255-261). The 3′ NIR is predicted to form astable secondary structure which is essential for HCV growth in chimpsand is believed to function in the initiation and regulation of viralRNA replication.

The NSSB protein (591 amino acids, 65 kDa) of HCV (Behrens, S. E. et al(1996) EMBO J. 15:12-22), encodes an RNA-dependent RNA polymerase (RdRp)activity and contains canonical motifs present in other RNA viralpolymerases. The NS5B protein is fairly well conserved bothintra-typically (˜95-98% amino acid (aa) identity across 1b isolates)and inter-typically (˜85% aa identity between genotype 1a and 1bisolates). The essentiality of the HCV NS5B RdRp activity for thegeneration of infectious progeny virions has been formally proven inchimpanzees (A. A. Kolykhalov et aL. (2000) Journal of Virology, 74(4),p.2046-2051). Thus, inhibition of NS5B RdRp activity (inhibition of RNAreplication) is predicted to cure HCV infection.

Based on the foregoing, there exists a significant need to identifysynthetic or biological compounds for their ability to inhibit HCV.

SUMMARY OF THE INVENTION

The present invention involves compounds represented hereinbelow,pharmaceutical compositions comprising such compounds and use of thecompounds in treating viral infection, especially HCV infection.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compounds of Formula (I):

wherein:

-   -   A represents OR¹, NR¹R², or R¹ wherein R¹ and R² are        independently selected from the group consisting of hydrogen,        C₁₋₆alkyl, aryl, heteroaryl, arylalkyl, and heteroarylalkyl; or        R¹ and R² together with the nitrogen atom to which they are        attached form a 5 or 6 membered saturated cyclic group;    -   B represents C(O)R³ wherein R³ is selected from the group        consisting of C₁₋₆alkyl, aryl, heteroaryl, arylalkyl, and        heteroarylalkyl;    -   D represents C₁₋₆alkyl, aryl, heteroaryl or heterocyclyl;    -   E represents OR¹, NR¹R², or R¹ wherein R¹ and R² are        independently selected from the group consisting of hydrogen,        C₁₋₆alkyl, aryl, heteroaryl arylalkyl or heteroarylalkyl; or R¹        and R² together with the nitrogen atom to which they are        attached form a 5 or 6 membered saturated cyclic group;    -   F represents hydrogen, C₁₋₆alkyl, aryl or heteroaryl; and    -   G represents hydrogen, C₁₋₆ alkyl, heterocyclylalkyl, arylalkyl        or heteroarylalkyl; and salts, solvates and esters thereof,        provided that when A is OR¹ then R¹ is other than tert-butyl.

As used herein, “alkyl” refers to an optionally substituted hydrocarbongroup. The alkyl hydrocarbon group may be linear, branched or cyclic,saturated or unsaturated. Where the alkyl hydrocarbon group is cyclic,it will be understood that there will be a minimum of 3 carbon atoms inthe group. Preferably, the group is saturated. Preferred alkyl moietiesare C₁₋₄alkyl. Optional subsituents include C₁₋₆alkyl, halo, OR⁴,C(O)NR⁵R⁶, C(O)R³, CO₂H, CO₂R³, NR⁵R⁶, NHC(O)R³, NHCO₂R³, NHC(O)NR¹R²,SO₂NR¹R², SO₂R³, nitro, cyano, oxo, and heterocyclyl. More preferably,the optional subsituents include C₁₋₆alkyl, halo, OR⁴, C(O)NR⁵R⁶, CO₂R³,NR⁵R⁶, NHC(O)R³, NHCO₂R³, NHC(O)NR¹R², SO₂NR¹R², SO₂R³, nitro, oxo, andheterocyclyl.

R⁴ represents hydrogen, C₁₋₆alkyl, arylalkyl, or heteroarylalkyl; R⁵ andR⁶ are independently selected from hydrogen, C₁₋₆alkyl, aryl andheteroaryl.

As used herein, “aryl” refers to an optionally substituted aromaticgroup with at least one ring having a conjugated pi-electron system,containing up to two conjugated or fused ring systems. “Aryl” includescarbocyclic aryl and biaryl groups, all of which may be optionallysubstituted. Preferred “aryl” moieties are unsubstituted,monosubstituted, disubstituted or trisubstituted phenyl. Preferred“aryl” substituents are selected from the group consisting of C₁₋₆alkyl,halo, OR⁴, C(O)NR⁵R⁶, C(O)R³, CO₂H, CO₂R³, NR⁵R⁶, NHC(O)R³, NHCO₂R³,NHC(O)NR¹R², SO₂NR¹R², SO₂R³, nitro, cyano, oxo, heterocyclyl, CF₃,pyridine, phenyl, and NO₂. More preferably, “aryl” substituents areselected from the group consisting of C₁₋₆alkyl, halo, OR⁴, C(O)NR⁵R⁶,CO₂R³, NR⁵R⁶, NHC(O)R³, NHCO₂R³, NHC(O)NR¹R², SO₂NR¹R², SO₂R³, nitro,oxo, heterocyclyl, OC₁₋₄alkyl, CF₃, pyridine, phenyl, and NO₂.

As used herein, “heteroaryl” refers to an optionally substituted, 5 or 6membered, aromatic group comprising one to four heteroatoms selectedfrom N, O and S, with at least one ring having a conjugated pi-electronsystem, containing up to two conjugated or fused ring systems. Preferred“heteroaryl” moieties are unsubstituted, monosubstituted, disubstitutedor trisubstituted thienyl and thiazolyl. Preferred “heteroaryl”substituents are selected from the group consisting of C₁₋₆alkyl, halo,OR⁸, C(O)NR⁶R⁷, C(O)R³, CO₂H, CO₂R³, NR⁶R⁷, NHC(O)R³, NHCO₂R³,NHC(O)NR¹R², SO₂NR¹R², SO₂R³, nitro, cyano, oxo, heterocyclyl, CF₃,pyridine, phenyl, and NO₂. More preferred “heteroaryl” substituents areselected from the group consisting of C₁₋₆alkyl, halo, OR⁴, C(O)NR⁵ ⁶,CO₂R³, NR⁵R⁶, NHC(O)R³, NHCO₂R³, NHC(O)NR¹R², SO₂NR¹R², SO₂R³, nitro,oxo, heterocyclyl, OC₁₋₄alkyl, CF₃, pyridine, phenyl, and NO₂.

As used herein, “heterocyclic” and “heterocyclyl” refer to an optionallysubstituted, 5 or 6 membered, saturated cyclic hydrocarbon groupcontaining one to four, preferably 1 or 2, heteroatoms selected from N,optionally substituted by hydrogen, C₁₋₆alkyl, C(O)R³, SO₂R³, aryl orheteroaryl; O; and S, optionally substituted by one or two oxygen atoms.

It will be appreciated that the compounds of the present invention maycontain one or more asymmetric carbon atoms and may exist in racemic andoptically active forms. All of these compounds and diastereoisomers arecontemplated to be within the scope of the present invention.

Preferably, A is OR¹ where R¹ is hydrogen;

Preferably, when B represents C(O)R³, R³ is aryl or heteroaryl; morepreferably, R³ is phenyl; especially preferred is R³ represents phenylsubstituted at least in the para-position by tert-butyl; most preferredis R³ represents phenyl substituted in the para-position by tert-butyland optionally further substituted, preferably meta-substituted, bymethyl, ethyl, methoxy, ethoxy, or halo, preferably halo;

Preferably, D is selected from the group consisting of C₁₋₆alkyl, aryland heteroaryl; more preferably D is heteroaryl; most preferably D is1,3-thiazolyl;

Preferably, E is OR¹ where R¹ is hydrogen; or NR¹R² where R¹ and R² areindependently selected from H, C₁₋₆alkyl, or arylalkyl;

Preferably, F is hydrogen or C₁₋₆alkyl; more preferably F is hydrogen;

Preferably, G is selected from the group consisting of C₁₋₆alkyl,arylalkyl and heteroarylalkyl; more preferably G is C₁₋₆alkyl.

It is to be understood that the present invention covers allcombinations of suitable, convenient and preferred groups describedherein.

Preferred compounds useful in the present invention are selected fromthe group consisting of:

-   -   rel-(2S,5R)-1-(4-tert-butylbenzoyl)-2-isobutyl-5-(1,3-thiazol-2-yl)-2,5-dihydro-1H-pyrrole-2,4dicarboxylic        acid;    -   rel-(2S,5R)-1-(4-tert-butylbenzoyl)-2-isobutyl-3-methyl-5-(1,3-thiazol-2-yl)-2,5-dihydro-1H-pyrrole-2,4-dicarboxylic        acid;    -   rel-(2S,5R)-1-(4-tert-butylbenzoyl)-4carbamoyl-2-isobutyl-5-(1,3-thiazol-2,5-dihydro-1H-pyrrole-2-carboxylic        acid;    -   rel-(2S,5R)-1-(4-tert-butylbenzoyl)-4{[(-2-carboxyethyl)amino]carbonyl}-2-isobutyl-5-(1,3-thiazol-2-yl)-2,5-dihydro-1H-pyrrole-2-carboxylic        acid;    -   rel-(2S,5R)-1-(4-tert-butylbenzoyl)-4-{[(3-carboxamidoethyl)amino]carbonyl}-2-isobutyl-5-(1,3-thiazol-2-yl)-2,5dihydro-1H-pyrrole-2-carboxylic        acid;    -   rel-(2S,5R)-1-(4-tert-butylbenzoyl)-4-{[(2-carboxymethyl)amino]carbonyl}-2-isobutyl-5-(1,3-thiazol-2-yl)-2,5-dihydro-1H-pyrrole-2-carboxylic        acid;    -   rel-(2S,5R)-1-(4-tert-butylbenzoyl)-4[(isobutylamino)carbonyl]-2-isobutyl-5-(1,3-thiazol-2-yl)-2,5-dihydro-1H-pyrrole-2-carboxylic        acid;    -   rel-(2S,5R)-1-(4-tert-butylbenzoyl)-4-[(benzylamino)carbonyl]-2-isobutyl-5-(1,3-thiazol-2-yl)-2,5-dihydro-1H-pyrrole-2-carboxylic        acid;    -   rel-(2S,5R)-1-(4-tert-butylbenzoyl)-4-{[(cyclohexylmethyl)amino]carbonyl}-2-isobutyl-5-(1,3-thiazol-2-yl)-2,5dihydro-1H-pyrrole-2-carboxylic        acid;    -   rel-(2S,5R)-1-(4-tert-butylbenzoyl)-4-{[(cyanomethyl)amino]carbonyl}-2-isobutyl-5-(1,3-thiazol-2-yl)-2,5dihydro-1H-pyrrole-2-carboxylic        acid;    -   rel-(2S,5R)-1-(3-bromo-4-tert-butylbenzoyl)-2-isobutyl-5-(1,3-thiazol-2-yl)-2,5-dihydro-1H-pyrrole-2,4-dicarboxylic        acid;    -   rel-(2S,5R)-1-(3-bromo-4-tert-butylbenzoyl)-4-carbamoyl-2-isobutyl-5-(1,3-thiazol-2-yl)-2,5dihydro-1H-pyrrole-2-carboxylic        acid;    -   and salts, solvates, esters and individual enantiomers thereof.

Also included in the present invention are pharmaceutically acceptablesalt complexes. The present invention also covers the physiologicallyacceptable salts of the compounds of formula (I). Suitablephysiologically acceptable salts of the compounds of formula (I) includeacid salts, for example sodium, potassium, calcium, magnesium andtetraalkylammonium and the like, or mono- or di-basic salts with theappropriate acid for example organic carboxylic acids such as acetic,lactic, tartaric, malic, isethionic, lactobionic and succinic acids;organic sulfonic acids such as methanesulfonic, ethanesulfonic,benzenesulfonic and p-toluenesulfonic acids and inorganic acids such ashydrochloric, sulfuric, phosphoric and sulfamic acids and the like.

The present invention also relates to solvates of the compounds ofFormula (I), for example hydrates.

The present invention also relates to pharmaceutically acceptable estersof the compounds of Formula (I), for example carboxylic acid esters—COOR, in which R is selected from straight or branched chain alkyl, forexample n-propyl, n-butyl, alkoxyalkyl (e.g. methoxymethyl), aralkyl(e.g.benzyl), aryloxyalkyl (e.g. phenoxymethyl), aryl (e.g. phenyloptionally substituted by halogen, C₁₋₄alkyl or C₁₋₄alkoxy or amino).Unless otherwise specified, any alkyl moiety present in such esterspreferably contains 1 to 18 carbon atoms, particularly 1 to 4 carbonatoms. Any aryl moiety present in such esters preferably comprises aphenyl group.

It will further be appreciated that certain compounds of the presentinvention may exist in different tautomeric forms. All tautomers arecontemplated to be within the scope of the present invention.

Compounds of Formula (I) may be prepared by reaction of a compound ofFormula (II)

in which A, D, E, F and G are as defined above for Formula (I); with asuitable acylating agent, for example R³C(O)-hal, wherein hal is a haloatom, preferably chloro or bromo. Preferably the reaction is carried outin a suitable solvent, for example dichloromethane or chloroform, in thepresence of a suitable base, for example triethylamine.

Compounds of Formula (II) may be prepared by reaction of a compound ofFormula (III)

wherein A, D and G are as defined for Formula (I) above; with a compoundof Formula (IV)

wherein E and F are as defined for Formula (I). Preferably, the reactionis carried out in a suitable solvent, for example TBF or acetonitrile,optionally in the presence of a Lewis acid catalyst, such as lithiumbromide or silver acetate, and a base, such as triethylamine,1,8-diazabicyclo[5,4,0]undec-7-ene (DBU) or tetramethyl guanidine.Alternatively, the reaction is carried out in a suitable solvent, forexample THF or acetonitrile, in the presence of an acid, such as aceticacid, or the reaction may be carried out by heating compounds of Formula(III) and Formula (IV) in a suitable solvent, for example toluene,xylene or acetonitrile in the absence of a catalyst.

Compounds of Formula (III) and (IV) are known in the art or may beprepared by standard literature procedures.

It will be appreciated that racemic compounds of Formula (I) and (II)may be optionally resolved into their individual enantiomers. Suchresolutions may conveniently be accomplished by standard methods knownin the art For example, a racemic compound of

Formula (I) and (II) may be resolved by chiral preparative HPLC.Alternatively, racemic compounds of Formula (I) and (II) may be resolvedby standard diastereoisomeric salt formation with a chiral acid or basereagent as appropriate. Such techniques are well established in the art.

With appropriate manipulation and protection of any chemicalfunctionality, synthesis of the remaining compounds of Formula (I) isaccomplished by methods analogous to those above and to those describedin the Experimental section. Example protecting groups can be found, butare not restricted to, those found in T. W. Greene and P. G. M. Wuts‘Protective Groups in Organic Synthesis’, 3^(rd) Ed (1999), J Wiley andSons. It will be appreciated that with appropriate manipulation, acompound of Formula (I) may be interconverted to a compound of Formula(I) with a different D group by methods well known in the art.

EXAMPLES Intermediate 12-[N-(1,3-Thiazol-2-yhnethylene)amino]-4-methyl-pentanoic acid,tert-butyl ester

A stirred mixture of 2-amino-4-methyl-pentanoic acid tert-butyl ester,hydrochloride (5.00 g, 22.34 mmol), 1,3-thiazole-2-carboxaldehyde (2.53g, 22.34 mmol) and triethylamine (3.10 mL, 22.3 mmol) in dichloromethane(60 mL) was heated under reflux under nitrogen for 19 hours. Thereaction mixture was allowed to cool to room temperature, washed twicewith water, dried over Na₂SO₄ and evaporated to give the title compoundas an oil.

¹H NMR (CDCl₃): δ 8.46 (s, 1H), 7.94 (d, 1H), 7.44 (dd, 1H), 4.07 (dd,1), 1.89-1.74 (m, 2H), 1.64-1.52 (m, 1H), 1.48 (s, 9H), 0.96 (d, 3H) and0.90 (d, 3H).

Intermediate 2rel-(2S,5R)-2-isobutyl-5-(1,3thiazol-2-yl)-2,5-dihydro-1H-pyrrole-2,4-dicarboxylicacid, 2-tert-butyl ester, 4-methyl ester

To a cooled (0° C.) stirred solution of Intermediate 1 (0.250 g, 0.885mmol) in anhydrous THF (5 mL) under nitrogen, was added triethylamine(0.123 mL, 1 eq.) followed by lithium bromide (77 mg, 1 eq.) andmethylpropiolate (0.08 mL, 1 eq.).

The mixture was stirred at 0° C. for 5 minutes, and then the ice/waterbath was removed and stirring was continued at ambient temperature for 2hours. Aqueous ammonium chloride was added with rapid stirring and theresulting mixture was extracted twice with ethyl acetate. The combinedorganic layers were dried over sodium sulphate and evaporated. Theresidue was purified by chromatography on silica gel usingcyclohexane-ethyl acetate (8:2 v/v) as eluent to provide the titlecompound as an oil.

Mass spec m/z calcd for (C₁₈H₂₆N₂O₄S+H)⁺: 367 Mass spec (electrospray)Found: (M+H)⁺ 367.

Intermediate 3rel-(2S,5R)-1-(4-tert-butylbenzoyl)-2-isobutyl-5-(1,3-thiazol-2-yl)-2,5-dihydro-1H-pyrrole-2,4-dicarboxylicacid, 2-tert-butyl ester, 4-methyl ester

To a stirred solution of Intermediate 2 (0.137 mg, 0.37 mmol) inanhydrous dichloromethane (5 mL) was added triethylamine (0.064 mL, 0.46mmol) and 4-tert-butylbenzoyl chloride (0.082 mL, 0.44 mmol). Thismixture was stirred and heated under reflux for 18 hours. The mixturewas then washed with water and extract with dichloromethane. The organicphase was dried (Na₂SO₄) and evaporated. The residue was purified bychromatography on silica gel using cyclohexane-ethyl acetate (6:1 v/v)as eluent to provide the title compound as a solid.

Mass spec m/z calcd for (C₂₉H₃₉N₂O₅S+H)⁺: 527. Mass spec (electrospray)Found: (M+H)⁺=527.

Intermediate 4rel-(2S,5R)-1-(3bromo-4-tert-butylbenzoyl)-2-isobutyl-5(1,3thiazol-2-yl)-2,5-dihydro-1H-pyrrole-2,4-dicarboxylicacid, 2-butyl ester, 4-methyl ester

Prepared in a similar manner to that described in Intermediate 3 usingIntermediate 2 and 3-bromo-4-tert-butylbenzoyl chloride. Chromatographyon silica gel using dichloromethane-ethyl acetate (50:1 v/v) as eluentprovided the title compound as a solid.

Mass spec m/z calcd for (C₂₉H₃₇BrN₂O₅S+H)⁺: 605/607 Mass spec(electrospray) Found: (M+H)⁺=605/607

Intermediate 5rel-(2S,5R)-1-(4-tert-butylbenzoyl)-2-isobutyl-5-(1,3-thiazol-2-yl)-2,5-dihydro-1H-pyrrole-2,4-dicarboxylicacid, 2-tert-butyl ester

To a solution of Intermediate 3 (64.5 mg, 0.12 mmol) in methanol (4 mL)was added a solution of 2M sodium hydroxide (0.122 mL) and the resultingmixture was stirred and heated under reflux for 20 hours. The solventwas evaporated and the residue was dissolved in dichloromethane (5 mL),acidified with hydrochloric acid (2M) and extracted withwater/dichloromethane. The organic phase was dried (Na₂SO₄) andevaporated to give the title compound as a solid.

Mass spec m/z calcd for (C₂₈H₃₆N₂O₅S+H)⁺: 513. Mass spec (electrospray)Found: (M+H)⁺=513.

Intermediate 6rel(2S,5R)-1-(3-bromo-4-tert-butylbenzoyl)-2-isobutyl-5-(1,3-thiazol-2-yl)-2,5-dihydro-1H-pyrrole-2,4-dicarboxylicacid, 2-tert-butyl ester

Prepared in a similar manner to that described for Intermediate 5 usingIntermediate 4.

Mass spec m/z calcd for (C₂₈H₃₅BrN₂O₅S+H)⁺: 591/593 Mass spec(electrospray) Found (M+H)⁺=591/593

Intermediate 7rel-(2S,5R)-2-isobutyl-3-methyl-5-(1,3-thiazol-2-yl)-2,5-dihydro-1H-pyrrole-2,4-dicarboxylicacid, 2-tert-butyl ester, 4-methyl ester

Intermediate 1 (500 mg, 1.77 mmol.) was dissolved in toluene (8 mL) andmethyl-2-butynoate (0.18 ml, 1.77 mmol) was added. The solution washeated under reflux over 72 hours and was then quenched with a solutionof saturated NH₄Cl (5 mL) and extracted with ethyl acetate. The combinedorganic layers were dried with Na₂SO₄ and concentrated. The residue waspurified by chromatography on silica gel using cyclohexane-ethyl acetate(8:2 v/v) as eluent, to afford the title compound.

Mass spec m/z calc. for (C₁₉H₂₈N₂O₄S+H)⁺: 381 Mass spec (electrospray)Found (M+H)⁺: 381

Intermediate 8rel-(2S,5R)-1-(4-tert-butylbenzoyl)-2-isobutyl-3-methyl-5-(1,3-thiazol-2-yl)-2,5-dihydro-1H-pyrrole-2,4-dicarboxylicacid, 2-tert-butyl ester, 4-methyl ester

In a similar manner to that described in Intermediate 3 and usingIntermediate 7, the title compound was prepared and purified by reversephase HPLC (ABZ column, 10 cm×21.2 cm×5 um, solvent A: 950:50:0.05acetonitrile:water:formic acid. solvent B: 0.1% aqueous formic acid.Gradient from 40% solvent A to 100% solvent A) and obtained as a solid.

Mass spec m/z calc. for (C₃₀H₄₀O₅N₂S+H)⁺: 541 Mass spec (electrospray)Found: (M+H)⁺: 541

Intermediate 9rel-(2S,5R)-1-(4-tert-butylbenzoyl)-2-isobutyl-3-methyl-5-(1,3-thiazol)-2-yl)-2,5dihydro-1H-pyrrole-2,4-dicarboxylicacid, 2-tert-butyl ester

In a similar manner to that described for Intermediate 4 and usingIntermediate 8, the title compound was prepared as a solid.

Mass spec m/z calc. for (C₂₉H₃₈O₅N₂S+H)⁺: 527 Mass spec (electrospray)Found: (M+H)⁺: 527

Intermediate 10rel-(2S,5R)-1-(4-tert-butylbenzoyl)-4-carbamoyl-2-isobutyl-5-(1,3thiazol-2-yl)-2,5-dihydro-1H-pyrrole-2-carboxylicacid, 2-tert-butyl ester

To a solution of Intermediate 5 (40 mg, 0.08 mmol) dissolved in DMF(dimethylformamide) (5 mL) was added DIPEA (diisopropylethylamine) (27ml; 0.16 mmol, 2 eq), ammonium chloride (6 mg, 0.08 mmol) and HATU([O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium]hexafluorophosphate)coupling reagent (30 mg, 0.08 mol). The resulting mixture was stirred atroom temperature overnight The reaction was quenched with a saturatedsolution of NaHCO₃ (15 mL) and extracted with water and ethyl acetate.The combined organic layers were dried (Na₂SO₄) and evaporated to give acrude compound that was purified by reverse phase HPLC (ABZ column, 10cm×21.2 cm×5 um, solvent A: 950:50:0.05 acetonitrile:water:formic acid.solvent B: 0.1% aqueous formic acid. Gradient from 40% solvent A to 100%solvent A) thus affording the title compound.

Mass spec m/z calc. for (C₂₈H₃₇N₃O₄S+H)⁺: 512 Mass spec (electrospray)Found (M+H)⁺: 512.

Intermediates 11-17 were prepared in a similar manner from Intermediate5 using the appropriate reagent.

Intermediate 11rel-(2S,5R)-1-(4-tert-butylbenzoyl)-4-{[(3-tert-butoxycarbonylethyl)amino]carbonyl}-2-isobutyl-5-(1,3-thiazol-2-yl)-2,5-dihydro-1H-pyrrole-2-carboxylicacid, 2tert-butyl ester

Mass spec m/z calc. for (C₃₅H₄₉N₃O₆S+H)⁺: 640 Mass spec (electrospray)Found (M+H)⁺: 640.

Intermediate 12rel-(2S,5R)-1-(4-tert-butylbenzoyl)-4-{[(3-carboxamidoethyl)amino]carbonyl}-2-isobutyl-5-(1,3-thiazol-2-yl)-2,5dihydro-1H-pyrrole-2-carboxylicacid, 2-tert-butyl ester

Mass spec m/z calc. for (C₃₁H₄₂N₄O₅S+H)⁺: 583 Mass spec (electrospray)Found (M+H)⁺: 583

Intermediate 13rel-(2S,5R)-1-(4tert-butylbenzoyl)-4-}[(2-tert-butoxycarbonylmethyl)amino]carbonyl}-2-isobutyl-5-(1,3thiazol-2-yl)-2,5dihydro-1H-pyrrole-2-carboxylicacid, 2-tert-butyl ester

Mass spec m/z calc. for (C₃₄H₄₇N₃O₆S+H)⁺: 626 Mass spec (electrospray)Found (M+H)⁺: 626.

Intermediate 14rel-(2S,5R)-1-(4-tert-butylbenzoyl)-4-[(isobutylamino)carbonyl]-2-isobutyl-5-(1,3-thiazol-2-yl)-2,5-dihydro-1H-pyrrole-2-carboxylicacid, 2-tert-butyl ester

Mass spec m/z calc. for (C₃₂H₄₅N₃O₄S+H)₊: 568 Mass spec (electrospray)Found (M+H)⁺: 568.

Intermediate 15rel-(2S,5R)-1-(4-tert-butylbenzoyl)-4-[(benzylamino)carbonyl]-2-isobutyl-5-(1,3-thiazol-2-yl)-2,5-dihydro-1H-pyrrole-2-carboxylicacid, 2-tert-butyl ester

Mass spec m/z calc. for (C₃₅H₄₃N₃O₄S+H)⁺: 602 Mass spec (electrospray)Found (M+H)⁺: 602

Intermediate 16rel-(2S,5R)-1-(4-tert-butylbenzoyl)-4-{[(cyclohexylmethyl)amino]carbonyl}-2-isobutyl-5-(1,3-thiazol-2-yl)-2,5-dihydro-1H-pyrrole-2-carboxylicacid, 2-tert-butyl ester

Mass spec m/z calc. for (C₃₅H₄₉N₃O₄S+H)⁺: 608 Mass spec (electrospray)Found (M+H)⁺: 608.

Intermediate 17 rel-(2S,5)-1-(4-butylbenzoyl)-4-}[(cyanomethyl)amino]carbonyl}-2-isobutyl-5-(1,3-thiazol-2-yl)-2,5-dihydro-1H-pyrrole-2-carboxylicacid, 2-tert-butyl ester

Mass spec m/z calc. for (C₃₀H₃₈N₄O₄S+H)⁺: 551 Mass spec (electrospray)Found (M+H)⁺: 551.

Intermediate 18rel-(2S,5R)-1-(3bromo-4-tert-butylbenzoyl)-4-carbamoyl-2-isobutyl-5-(1,3-thiazol-2-yl)-2,5-dihydro-1H-pyrrole-2,4dicarboxylicacid, 2-tert-butyl ester

Prepared in a similar manner to that described for Intermediate 10, fromIntermediate 6.

Mass spec m/z calc. for (C₂₈H₃₆BrN₃O₄S+H)⁺: 590/592 Mass spec(electrospray) Found (M+H)⁺: 590/592.

Example 1rel-(2S,5R)-1-(4-tert-butylbenzoyl)-2-isobutyl-5-(1,3-thiazol-2-yl)-2,5-dihydro-1H-pyrrole-2,4dicarboxylicacid

To a solution of Intermediate 5 (44 mg, 8.85 mmol) in dichloromethane (5mL) was added trifluoroacetic acid (1 mL) and the solution stirred atambient temperature overnight The reaction mixture was evaporated andthe residue triturated with diethylether to give the title compound as asolid.

Mass spec m/z calc for (C₂₄H₂₈N₂O₅S+H)⁺: 457 Mass spec (electrospray)Found: (M+H)⁺457

Example 2rel-(2S,5R)-1-(4-tert-butylbenzoyl)-2-isobutyl-3-methyl-5-(1,3-thiazol-2-yl)-2,5-dihydro-1H-pyrrole-2,4-dicarboxylicacid

Using Intermediate 9 and in a similar manner to that described inExample 1, the title compound was prepared as a solid.

Mass spec m/z calc. for (C₂₅H₃₀N₂O₅S+H)⁺: 471 Mass spec (electrospray)Found: (M+H)⁺: 471

Example 3rel-(2S,5R)-1-(4tert-butylbenzoyl)-4-carbamoyl-2-isobutyl-5-(1,3-thiazol-2-yl)-2,5-dihydro-1H-pyrrole-2-carboxylicacid

Using Intermediate 10 and in a similar manner to that described inExample 1, the title compound was prepared as a solid.

Mass Spec m/z calc for (C₂₄H₂₉N₃O₄S+H)⁺: 456 Mass spec (electrospray)Found (M+H)⁺: 456.

Example 4rel-(2S,5R)-1-(4-tert-butylbenzoyl)-4-{[(2-carboxyethyl)amino]carbonyl}-2-isobutyl-5-(1,3-thiazol-2-yl)-2,5-dihydro-1H-pyrrole-2-carboxylicacid

A solution of Intermediate 11 (45 mg) in dichloromethane (1 mL) wastreated with trifluoroacetic acid (1 mL) and placed at room temperaturefor 5 hours. Volatile materials were removed and the residue wastriturated with ether to give the title compound as a solid.

Mass spec m/z calc for (C₂₇H₃₃N₃O₆S+H)⁺: 528 Mass spec (electrospray)Found (M+H)⁺: 528.

Examples 5-12 were prepared in a similar manner.

Example 5rel-(2S,5R)-1-(4-tert-butylbenzoyl)-4-{[(3-carboxamidoethyl)amino]carbonyl}-2-isobutyl-5-(1,3-thiazol-2-yl)-2,5-dihydro-1H-pyrrole-2-carboxylicacid

Prepared from Intermediate 12.

Mass spec m/z calc for (C₂₇H₃₄N₄O₅S+H)⁺: 527 Mass spec (electrospray)Found (+H)⁺: 527.

Example 6rel-(2S,5S)-1-(4-tert-butylbenzoyl)-4-{[(2-carboxymethyl)amino]carbonyl}-2-isobutyl-5-(1,3-thiazol-2-yl)-2,5-dihydro-1H-pyrrole-2-carboxylicacid

Prepared from Intermediate 13.

Mass spec m/z calc for (C₂₆H₃₁N₃O₆S+H)⁺: 514. Mass spec (electrospray)Found (M+H)⁺: 514.

Example 7rel-(2S,5R)-1-(4-tert-butylbenzoyl)-4-[isobutylamino)carbonyl]-2-isobutyl-5-(1,3-thiazol-2yl)-2,5-dihydro-1H-pyrrole-2-carboxylicacid

Prepared from Intermediate 14.

Mass spec m/z calc for (C₂₈H₃₇N₃O₄S+H)⁺: 512. Mass spec (electrospray)Found (M+H)⁺: 512.

Example 8rel-(2S,5R)-1-(4-tert-butylbenzoyl)-4-[(benzylamino)carbonyl]-2-isobutyl-5-(1,3-thiazol-2-yl)-2,5-dihydro-1H-pyrrole-2-carboxylicacid

Prepared from Intermediate 15.

Mass spec m/z calc for (C₃₁H₃₅N₃O₄S+H)⁺: 546 Mass spec (electrospray)Found (M+H)⁺: 546

Example 9rel-(2S,5R)-1-(4tert-butylbenzoyl)-4-{[(cyclohexylmethyl)amino]carbonyl}-2-isobutyl-5-(1,3-thiazol-2-yl)-2,5-dihydro-1H-pyrrole-2-carboxylicacid

Prepared from Intermediate 16.

Mass spec m/z calc for (C₃₁H₄₁N₃O₄S+H)⁺: 552 Mass spec (electrospray)Found (M+H)⁺: 552

Example 10rel-(2S,5R)-1-(4-tert-butylbenzoyl)-4-{[(cyanomethyl)amino]carbonyl}-2-isobutyl-5-(1,3-thiazol-2-yl)-2,5-dihydro-1H-pyrrole-2-carboxylicacid

Prepared from Intermediate 17.

Mass spec m/z calc for (C₂₆H₃₀N₄O₄S+H)⁺: 495 Mass spec (electrospray)Found (M+H)⁺: 495

Example 11rel-(2S,5R)-1-(3-bromo-4-tert-butylbenzoyl)-2-isobutyl-5-(1,3-thiazol-2-yl)-2,5-dihydro-1H-pyrrole-2,4dicarboxylicacid

Prepared from Intermediate 6.

Mass spec m/z calc for (C₂₄H₂₇BrN₂O₅S+H)⁺: 535/537 Mass spec(electrospray) Found (M+H)⁺: 535/537

Example 12rel-(2S,5R)-1-(3-bromo-4-tert-butylbenzoyl)-4-carbamoyl-2-isobutyl-5-(1,3-thiazol-2-yl)-2,5-dihydro-1H-pyrrole-2-carboxylicacid

Prepared from Intermediate 18.

Mass spec m/z calc for (C₂₄H₂₈BrN₃O₄S+H)⁺: 534/536 Mass spec(electrospray) Found (M+H)⁺: 534/536

The compounds according to the invention may be formulated foradministration in any convenient way, and the invention therefore alsoincludes within its scope pharmaceutical compositions for use intherapy, comprising a compound of formula (I) or a physiologicallyacceptable salt or solvate thereof in admixture with one or morephysiologically acceptable diluents or carriers.

The compounds of the present invention can be administered by differentroutes including intravenous, intraperitoneal, subcutaneous,intramuscular, oral, topical, transdermal, or transmucosaladministration. For systemic administration, oral administration ispreferred. For oral administration, for example, the compounds can beformulated into conventional oral dosage forms such as capsules, tabletsand liquid preparations such as syrups, elixirs and concentrated drops.

Alternatively, injection (parenteral administration) may be used, e.g.,intramuscular, intravenous, intraperitoneal, and subcutaneous. Forinjection, the compounds of the invention are formulated in liquidsolutions, preferably, in physiologically compatible buffers orsolutions, such as saline solution, Hank's solution, or Ringer'ssolution. In addition, the compounds may be formulated in solid form andredissolved or suspended immediately prior to use. Lyophilized forms canalso be produced.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art and include, for example,for transmucosal administration, bile salts and fusidic acidderivatives. In addition, detergents may be used to facilitatepermeation. Transmucosal administration, for example, may be throughnasal sprays, rectal suppositories, or vaginal suppositories.

For topical administration, the compounds of the invention can beformulated into ointments, salves, gels, or creams, as is generallyknown in the art.

The amounts of various compounds to be administered can be determined bystandard procedures taking into account factors such as the compound(IC₅₀) potency, (EC₅₀) efficacy, and the biological half-life (of thecompound), the age, size and weight of the patient, and the disease ordisorder associated with the patient The importance of these and otherfactors to be considered are known to those of ordinary skill in the art

Amounts administered also depend on the routes of administration and thedegree of oral bioavailability. For example, for compounds with low oralbioavailability, relatively higher doses will have to be administered.Oral administration is a preferred method of administration of thepresent compounds.

Preferably the composition is in unit dosage form. For oral application,for example, a tablet, or capsule may be administered, for nasalapplication, a metered aerosol dose may be administered, for transdermalapplication, a topical formulation or patch may be administered and fortransmucosal delivery, a buccal patch may be administered. In each case,dosing is such that the patient may administer a single dose.

Each dosage unit for oral administration contains suitably from 0.01 to500 mg/Kg, and preferably from 0.1 to 50 mg/Kg, of a compound of Formula(I) or a pharmaceutically acceptable salt thereof, calculated as thefree base. The daily dosage for parenteral, nasal, oral inhalation,transmucosal or transdermal routes contains suitably from 0.01 mg to 100mg/Kg, of a compound of Formula(I). A topical formulation containssuitably 0.01 to 5.0% of a compound of Formula (I). The activeingredient may be administered from 1 to 6 times per day, preferablyonce, sufficient to exhibit the desired activity, as is readily apparentto one skilled in the art.

Composition of Formula (I) and their pharmaceutically acceptable saltswhich are active when given orally can be formulated as syrups, tablets,capsules and lozenges. A syrup formulation will generally consist of asuspension or solution of the compound or salt in a liquid carrier forexample, ethanol, peanut oil, olive oil, glycerine or water with aflavoring or coloring agent. Where the composition is in the form of atablet, any pharmaceutical carrier routinely used for preparing solidformulations may be used. Examples of such carriers include magnesiumstearate, terra alba, talc, gelatin, acacia, stearic acid, starch,lactose and sucrose. Where the composition is in the form of a capsule,any routine encapsulation is suitable, for example using theaforementioned carriers in a hard gelatin capsule shell. Where thecomposition is in the form of a soft gelatin shell capsule anypharmaceutical carrier routinely used for preparing dispersions orsuspensions may be considered, for example aqueous gums, celluloses,silicates or oils, and are incorporated in a soft gelatin capsule shell.

Typical parenteral compositions consist of a solution or suspension of acompound or salt in a sterile aqueous or non-aqueous carrier optionallycontaining a parenterally acceptable oil, for example polyethyleneglycol, polyvinylpyrrolidone, lecithin, arachis oil or sesame oil.

Typical compositions for inhalation are in the form of a solution,suspension or emulsion that may be administered as a dry powder or inthe form of an aerosol using a conventional propellant such asdichlorodifluoromethane or trichlorofluoromethane.

A typical suppository formulation comprises a compound of Formula (I) ora pharmaceutically acceptable salt thereof which is active whenadministered in this way, with a binding and/or lubricating agent, forexample polymeric glycols, gelatins, cocoa-butter or other low meltingvegetable waxes or fats or their synthetic analogs.

Typical dermal and transdermal formulations comprise a conventionalaqueous or non-aqueous vehicle, for example a cream, ointment, lotion orpaste or are in the form of a medicated plaster, patch or membrane.

No unacceptable toxological effects are expected when compounds of thepresent invention are administered in accordance with the presentinvention.

Assay

The potential for compounds of the invention to inhibit NS5B wildtypeHCV polymerase activity may be demonstrated, for example, using thefollowing in vitro assay:

In Vitro Detection of Inhibitors of HCV RNA-Dependent RNA PolymeraseActivity

Incorporation of [³H]-UMP into RNA was followed by absorption of the RNApolymer onto a DEAE glass fibre filter. A synthetic template consistingof 16 mer oligoU hybridised to polyrA (10:1 w/w) was used as ahomopolymer substrate.

Reaction Conditions were 22 μM [³H]-UTP (0.75 Ci/mmol), 1mM-Dithiothreitol, 3.2 mM-MgCl₂, 20 mM-Tris-HCl, pH 7.0, 10 μg/mlpolyA-oligoU, and 90 mM-NaCl. Note that 50 mM-NaCl is added with theenzyme.

HCV RNA Polymerase (Recombinant full-length NS5B (Lohmann et al, J.Virol. 71 (11), 1997, 8416 ‘Biochemical properties of hepatitis C virusNS5B RNA-dependent RNA polymerase and identification of amino acidsequence motifs essential for enzymatic activity’) expressed inbaculovirus and purified to homogeneity) was diluted to about 50 μgprotein/mL (dependent on specific activity) in 50 mM-Hepes, pH 7.0,0.5M-NaCl, 20%-Glycerol, 0.05%-Triton X-100, 5 mM-Dithiothreitol, 0.1mM-EDTA.

5× Concentrated Buffer minx was prepared using 1M-Tris-HCl (pH 7.0, 1mL), 1M-MgCl₂ (0.16 mL), 1M-Dithiothreitol (0.05 mL), 5M-NaCl (0.4 mL),and Water (8.4 mL), Total 10 mL.

Substrate Mix was prepared using 5× Concentrated Buffer mix (12 μL),[³H]-UTP (1 μCi/μL; 21.7 μM, 1 μL), 22 μM-UTP (100 μM, 13.2 μL), 10μg/mL polyA-oligoU (100 μg/mL, 6 μL), and Water (12.8 μL), Total 45 μL.

The Assay was set up using Substrate Mix (45 μL), compound (10 μL), andDiluted Enzyme (added last to start reaction) (5 μL), Total 60 μL. Thereaction was performed in a U-bottomed, clear, 96-well plate. Thereaction was mixed on a plate-shaker, after addition of the Enzyme, andincubated for 2 h at 22° C. After this time, the reaction was stopped byaddition of 25 μL of 100 mM-EDTA.

A DEAE Filtermat (Part No. 1205-405 from Pharmacia) was pre-washed inwater and alcohol and dried. 2×20 μL of the Stopped Assay Mix wasspotted onto a square of the DEAE Filtermat. The DEAE Filtermat waswashed for 2×5 min in SSC buffer (0.3M-NaCl, 30 mM-Na Citrate) followedby 2×2 min in water and 1×1 min in alcohol. The Filtermat was dried andsealed in a bag together with 10 mL of OptiScint HiSafe scintillationfluid. The radioactivity present on the filtermat was detected byscintillation counting on a Wallac 1205 Betaplate counter. Aftersubtraction of background levels without enzyme, any reduction in theamount of radioactivity incorporated in the presence of a compound,compared to that in the absence, was taken as a measure of the level ofinhibition. Ten concentrations of compounds were tested in two- orthreefold dilutions. From the counts, percentage of inhibition athighest concentration tested or IC₅₀s for the compounds were calculatedusing Grafit3 or Grafit4 software packages.

The exemplified compounds had an IC₅₀ of <50 μm. Accordingly, thecompounds of the invention are of potential therapeutic benefit in thetreatment and prophylaxis of HCV. Preferred compounds had an IC₅₀ of <5μM.

Thus, there is provided as a further aspect of the present invention acompound of formula (I) or a physiologically acceptable salt or solvatethereof for use in human or veterinary medical therapy, particularly usein the treatment and/or prophylaxis of a viral infection, particularlyHCV infection.

It will be appreciated that reference herein to treatment includes, butis not limited to prevention, retardation, prophylaxis, therapy and cureof the disease. It will further be appreciated that references herein totreatment or prophylaxis of HCV infection includes treatment orprophylaxis of HCV-associated disease such as liver fibrosis, cirrhosisand hepatocellular carcinoma.

According to another aspect of the invention, there is provided the useof a compound of formula (I) or a physiologically acceptable salt orsolvate thereof for the manufacture of a medicament for the treatmentand/or prophylaxis of viral infection, particularly HCV infection.

In a further or alternative aspect there is provided a method for thetreatment of a human or animal subject with viral infection,particularly HCV infection, which method comprises administering to saidhuman or animal subject an effective amount of a compound of formula (I)or a physiologically acceptable salt or solvate thereof.

The pharmaceutical compositions according to the invention may also beused in combination with other therapeutic agents, for example immunetherapies (eg. interferon), therapeutic vaccines, antifibrotic agents,anti-inflammatory agents such as corticosteroids or NSAIDs,bronchodilators such as beta-2 adrenergic agonists and xanthines (e.g.theophylline), mucolytic agents, anti-muscarinics, anti-leukotrienes,inhibitors of cell adhesion (e.g. ICAM antagonists), anti-oxidants (egN-acetylcysteine), cytoline agonists, cytokine antagonists, lungsurfactants and/or antimicrobial and anti-viral agents (eg ribavirin andamantidine). The compositions according to the invention may also beused in combination with gene replacement therapy.

The invention thus provides, in a further aspect, a combinationcomprising a compound of formula (I) or a physiologically acceptablesalt or solvate thereof together with another therapeutically activeagent.

The combinations referred to above may conveniently be presented for usein the form of a pharmaceutical formulation and thus pharmaceuticalformulations comprising a combination as defined above together with apharmaceutically acceptable carrier thereof represent a further aspectof the invention. The individual components of such combinations may beadministered either sequentially or simultaneously in separate orcombined pharmaceutical formulations. Appropriate doses of knowntherapeutic agents will be readily appreciated by those skilled in theart.

All publications, including but not limited to patents and patentapplications cited in this specification are herein incorporated byreference as if each individual publication were specifically andindividually indicated to be incorporated by reference as though fullyset forth.

1. A compound of formula (I)

wherein: A represents OR¹, NR¹R², or R¹ wherein R¹ and R² areindependently selected from the group consisting of hydrogen, C₁₋₆alkyl,aryl, heteroaryl, arylalkyl, and heteroarylalkyl; or R¹ and R² togetherwith the nitrogen atom to which they are attached form a 5 or 6 memberedsaturated cyclic group; B represents C(O)R³ wherein R³ is selected fromthe group consisting of C₁₋₆alkyl, aryl, heteroaryl, arylalkyl, andheteroarylalkyl; D represents C₁₋₆alkyl, aryl, heteroaryl orheterocyclyl; E represents OR¹, NR¹R², or R¹ wherein R¹ and R² areindependently selected from the group consisting of hydrogen, C₁₋₆alkyl,aryl, heteroaryl, arylalkyl or heteroarylalkyl; or R¹ and R² togetherwith the nitrogen atom to which they are attached form a 5 or 6 memberedsaturated cyclic group; F represents hydrogen, C₁₋₆alkyl, aryl orheteroaryl; and G represents hydrogen, C₁₋₆alkyl, heterocyclylalkyl,arylalkyl or heteroarylalkyl; and salts, solvates and esters thereof,provided that when A is OR¹ then R¹ is other than tert-butyl.
 2. Acompound of Formula (I) as claimed in claim 1 selected from the groupconsisting of:rel-(2S,5R)-1-(4-tert-butylbenzoyl)-2-isobutyl-5-(1,3-thiazol-2-yl)-2,5-dihydro-1H-pyyrole-2,4-dicarboxylicacid;rel-(2S,5R)-1-(4-tert-butylbenzoyl)-2-isobutyl-3-methyl-5-(1,3-thiazol-2-yl)-2,5-dihydro-1H-pyrrole-2,4-dicarboxylicacid;rel-(2S,5R)-1-(4-tert-butylbenzoyl)-4-carbamoyl-2-isobutyl-5-(1,3-thiazol-2-yl)-2,5-dihydro-1H-pyrrole-2-carboxylicacid;rel-(2S,5R)-1-(4-tert-butylbenzoyl)-4-{[(2-carboxyethyl)amino]carbonyl}-2-isobutyl-5-(1,3-thiazol-2-yl)-2,5-dihydro-1H-pyrrole-2-carboxylicacid;rel-(2S,5R)-1-(4-tert-butylbenzoyl)-4-{[(3-carboxamidoethyl)amino]carbonyl}-2-isobutyl-5-(1,3-thiazol-2-yl)-2,5-dihydro-1H-pyrrole-2-carboxylicacid;rel-(2S,5R)-1-(4-tert-butylbenzoyl)-4{[(2-carboxymethyl)amino]carbonyl}-2-isobutyl-5-(1,3-thiazol-2-yl)-2,5-dihydro-1H-pyrrole-2-carboxylicacid;rel-(2S,5R)-1-(4-tert-butylbenzoyl)-4-[(isobutylamino)carbonyl]-2-isobutyl-5-(1,3-thiazol-2-yl)-2,5-dihydro-1H-pyrrole-2-carboxylicacid;rel-(2S,5R)-1-(4-tert-butylbenzoyl)-4-[(benzylamino)carbonyl]-2-isobutyl-5-(1,3-thiazol-2-yl)-2,5-dihydro-1H-pyrrole-2-carboxylicacid;rel-(2S,5R)-1-(4-tert-butylbenzoyl)-4-{[(cyclohexylmethyl)amino]carbonyl}-2-isobutyl-5-(1,3-thiazol-2-yl)-2,5-dihydro-1H-pyrrole-2-carboxylicacid;rel-(2S,5R)-1-(4-tert-butylbenzoyl)-4-{[(cyanomethyl)amino]carbonyl}-2-isobutyl-5-(1,3-thiazol-2-yl)-2,5-dihydro-1H-pyrrole-2-carboxylicacid;rel-(2S,5R)-1-(3-bromo-4-tert-butylbenzoyl)-2-isobutyl-5-(1,3-thiazol-2-yl)-2,5-dihydro-1H-pyrrole-2,4-dicarboxylicacid; andrel-(2S,5R)-1-(3-bromo-4-tert-butylbenzoyl)-4-carbamoyl-2-isobutyl-5-(1,3-thiazol-2-yl)-2,5-dihydro-1H-pyrrole-2-carboxylicacid; salts, solvates, esters and individual enantiomers thereof.
 3. Apharmaceutical formulation comprising a compound of Formula (I) asclaimed in claim 1 in conjunction with a pharmaceutically acceptablediluent or carrier therefor.
 4. A method of treating or preventing viralinfection which comprises administering to a subject in need thereof, aneffective amount of a compound as claimed in claim
 1. 5. A method asclaimed in claim 4 which involves inhibiting HCV.
 6. A method as claimedin claim 4 in which the compound is administered in oral dosage form. 7.(canceled)
 8. (canceled)
 9. (canceled)
 10. (canceled)
 11. (canceled) 12.A process for the preparation of a compound of Formula (I) as claimed inclaim 1, comprising reaction of a compound of Formula (II)

in which A, D, E, F and G are as defined above for Formula (I); with anacylating agent.
 13. A compound as claimed in claim 1, wherein Brepresents C(O)R³ and R³ represents phenyl substituted in thepara-position by tert-butyl and optionally further substituted bymethyl, ethyl, methoxy, ethoxy, or halo.